Humanized antibodies that sequester abeta peptide

ABSTRACT

A method to treat conditions characterized by formation of amyloid plaques both prophylactically and therapeutically is described. The method employs humanized antibodies which sequester soluble Aβ peptide from human biological fluids or which preferably specifically bind an epitope contained within position 13–28 of the amyloid beta peptide Aβ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT patent applicationPCT/US01/06191, filed Feb. 26, 2001, which was published in English anddesignated the United States and which claims the priority of U.S.provisional applications 60/184,601, filed Feb. 24, 2000, 60/256,465filed Dec. 8, 2000, and 60/254,498, filed Dec. 8, 2000. The contents ofeach of these applications are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to humanized antibodies that bind to an epitopebetween amino acids 13 and 28 of the Aβ peptide and to preventive andtherapeutic treatment of conditions associated with beta amyloid, suchas Alzheimer's disease, Down's syndrome, and cerebral amyloidangiopathey. More specifically, it concerns use of humanized monoclonalantibodies to sequester amyloid beta (Aβ) peptide in plasma, brain, andcerebrospinal fluid to prevent accumulation or to reverse deposition ofthe Aβ peptide within the brain and in the cerebrovasculature and toimprove cognition.

BACKGROUND ART

A number of symptomologies which result in cognitive deficits, stroke,brain hemorrhage, and general mental debilitation appear to beassociated with neuritic and cerebrovascular plaques in the braincontaining the amyloid beta peptide (Aβ). Among these conditions areboth pre-clinical and clinical Alzheimer's disease, Down's syndrome, andpre-clinical and clinical cerebral amyloid angiopathy (CAA). The amyloidplaques are formed from amyloid beta peptides. These peptides circulatein the blood and in the cerebrospinal fluid (CSF), typically incomplexed form with lipoproteins. The Aβ peptide in circulating form iscomposed of 39–43 amino acids (mostly 40 or 42 amino acids) resultingfrom the cleavage of a common precursor protein, amyloid precursorprotein, often designated APP. Some forms of soluble Aβ are themselvesneurotoxic and may determine the severity of neurodegeneration and/orcognitive decline (McLean, C. A., et al., Ann. Neurol. (1999)46:860–866; Lambert, M. P., et al. (1998) 95:6448–6453; Naslund, J., J.Am. Med. Assoc. (2000) 283:1571).

Evidence suggests that Aβ can be transported back and forth betweenbrain and the blood (Ghersi-Egea, J-F., et al., J. Neurochem. (1996)67:880–883; Zlokovic, B. V., et al, Biochem. Biophys. Res. Comm. (1993)67:1034–1040; Shibata M, et al., J. Clin. Invest. (2000) 106:1489–1499).Further Aβ in plaques is in an equilibrium with soluble Aβ in the brainand blood (Kawarabayashi T, et al., J. Neurosci. (2001) 21:372–381).

As described in PCT application US00/35681 and U.S. Ser. No. 09/753,130(now U.S. Pat. No. 6,465,195, issued Oct. 15, 2000) both incorporatedherein by reference, total circulating levels of Aβ peptide in OSE aresimilar in normal individuals and individuals predisposed to exhibit thesymptoms of Alzheimer's. However, Aβ₄₂ levels are lower on average inindividuals with Alzheimer's disease (Nitsch, R. M., et al., Ann.Neurol. (1995) 37:512–518). It is known that Aβ₄₂ is more prone toaggregate than is Aβ₄₀, and when this happens, adverse consequences suchas Aβ deposition in amyloid plaques, conversion of Aβ to toxic solubleforms, nerve cell damage, and behavioral impairment such as dementiasensue (Golde, T. E., et al., Biochem. Biophys. Acta. (2000)1502:172–187).

Methods to induce an immune response to reduce amyloid deposits aredescribed in PCT publication WO99/27944 published Jun. 10, 1999. Thedescription postulates that full-length aggregated Aβ peptide would be auseful immunogen. Administration of a Aβ fragment (amino acids 13–28)conjugated to sheep anti-mouse IgG caused no change in cortex amyloidburden, and only one in nine animals that received injections of the Aβ13–28 fragment-conjugate showed any lymphoproliferation in response toAβ₄₀. The application also indicates that antibodies that specificallybind to Aβ peptide could be used as therapeutic agents. However, thisappears to be speculation since the supporting data reflect protocolsthat involve active immunization using, for example, Aβ₄₂. The peptidesare supplied using adjuvants and antibody titers formed from theimmunization, as well as levels of Aβ peptide and of the precursorpeptide, are determined. The publication strongly suggests that Aβplaque must be reduced in order to alleviate Alzheimer's symptoms, andthat cell-mediated processes are required for successful reduction of Aβplaque.

WO 99/60024, published 25 Nov. 1999, is directed to methods for amyloidremoval using anti-amyloid antibodies. The mechanism, however, is statedto utilize the ability of anti-Aβ antibodies to bind to pre-formedamyloid deposits (i.e., plaques) and result in subsequent localmicroglial clearance of localized plaques. This mechanism was not provedin vivo. This publication further states that to be effective against Aβplaques, anti-Aβ antibodies must gain access to the brain parenchyma andcross the blood brain barrier.

Several PCT applications that relate to attempts to control amyloidplaques were published on 7 Dec. 2000. WO 00/72880 describes significantreduction in plaque in cortex and hippocampus in a transgenic mousemodel of Alzheimer's disease when treated using N-terminal fragments ofAβ peptides and antibodies that bind to them, but not when treated withthe Aβ 13–28 fragment conjugated to sheep anti-mouse IgG or with anantibody against the 13–28 fragment, antibody 266. The N-terminaldirected antibodies were asserted to cross the blood-brain barrier andto induce phagocytosis of amyloid plaques in in vitro studies.

WO 00/72876 has virtually the same disclosure as WO 00/72880 and isdirected to immunization with the amyloid fibril components themselves.

WO 00/77178 describes antibodies that were designed to catalyze thehydrolysis of β-amyloid, including antibodies raised against a mixtureof the phenylalanine statine transition compounds Cys-Aβ₁₀₋₂₅, statinePhe₁₉-Phe₂₀ and Cys-Ap₁₀₋₂₅ statine Phe₂₀-Ala₂₁ and antibodies raisedagainst Aβ₁₀₋₂₅ having a reduced amide bond between Phe₁₉ and Phe₂₀.This document mentions sequestering of Aβ, but this is speculationbecause it gives no evidence of such sequestering. Further, the documentprovides no in vivo evidence that administration of antibodies causesefflux of Aβ from the central nervous system, interferes with plaqueformation, reduces plaque burden, forms complexes between the antibodiesand Aβ in tissue samples, or affects cognition.

It has been shown that one pathway for Aβ metabolism is via transportfrom CNS to the plasma (Zlokovic, B. V., et al., Proc. Natl. Acad. Sci(USA) (1996) 93:4229–4234; Ghersi-Egea, J-F., et al., J. Neurochem.(1996) 67:880–883). Additionally, it has been shown that Aβ in plasmacan cross the blood-brain-barrier and enter the brain (Zlokovic, B. V.,et al., Biochem. Biophys. Res. Comm. (1993) 67:1034–1040). It has alsobeen shown that administration of certain polyclonal and monoclonal Aβantibodies decreases Aβ deposition in amyloid plaques in the APP^(V717F)transgenic mouse model of Alzheimer's disease (Bard, F., et al., NatureMed. (2000) 6:916–919); however, this was said to be due to certainanti-Aβ antibodies crossing the blood-brain-barrier stimulatingphagocytose of amyloid plaques by microglial cells. In Bard'sexperiments, assays of brain slices ex vivo showed that the presence ofadded Aβ antibody, along with exogenously added microglia, inducedphagocytosis of Aβ, resulting in removal of Aβ deposits.

The levels of both soluble Aβ₄₀ and Aβ₄₂ in CSF and blood can readily bedetected using standardized assays using antibodies directed againstepitopes along the Aβ chain. Such assays have been reported, forexample, in U.S. Pat. Nos. 5,766,846; 5,837,672; and 5,593,846. Thesepatents describe the production of murine monoclonal antibodies to thecentral domain of the Aβ peptide, and these were reported to haveepitopes around and including positions 16 and 17. Antibodies directedagainst the N-terminal region were described as well. Several monoclonalantibodies were asserted to immunoreact with positions 13–28 of the Aβpeptide; these did not bind to a peptide representing positions 17–28,thus, according to the cited patents, establishing that it is thisregion, including positions 16–17 (the α-secretase site) that was thetarget of these antibodies. Among antibodies known to bind between aminoacids 13 and 28 of Aβ are mouse antibodies 266, 4G8, and 1C2.

We have now unexpectedly found that administration of the 266 antibodyvery quickly and almost completely restores cognition (object memory) in24-month old hemizygous transgenic mice (APP^(V717F)). Yet, the antibodydoes not have the properties that the art teaches are required for anantibody to be effective in treating Alzheimer's disease, Down'ssyndrome, and other conditions related to the Aβ peptide. To our furthersurprise, we observed that antibodies that bind Aβ between positions 13and 28 (266 and 4G8) are capable of sequestering soluble forms of Aβfrom their bound, circulating forms in the blood, and that peripheraladministration of antibody 266 results in rapid efflux of relativelylarge quantities of Aβ peptide from the CNS into the plasma. Thisresults in altered clearance of soluble Aβ, prevention of plaqueformation, and, most surprisingly, improvement in cognition, evenwithout necessarily reducing Aβ amyloid plaque burden, crossing theblood brain barrier to any significant extent, decorating plaque,activating cellular mechanisms, or binding with great affinity toaggregated Aβ.

DISCLOSURE OF THE INVENTION

The invention provides humanized antibodies, or fragments thereof, thatpositively affect cognition in diseases and conditions where Aβ may beinvolved, such as clinical or pre-clinical Alzheimer's disease, Down'ssyndrome, and clinical or pre-clinical cerebral amyloid angiopathy. Theantibodies or fragments thereof need not cross the blood-brain barrier,decorate amyloid plaque, activate cellular responses, or evennecessarily reduce amyloid plaque burden. In another aspect, thisinvention provides humanized antibodies and fragments thereof thatsequester Aβ peptide from its bound, circulating form in blood, andalter clearance of soluble and bound forms of Aβ in central nervoussystem and plasma. In another aspect, this invention provides humanizedantibodies and fragments thereof, wherein the humanized antibodiesspecifically bind to an epitope between amino acids 13 and 28 of the Aβmolecule. In another aspect, the invention provides humanized antibodiesand fragments thereof, wherein the CDR are derived from mouse monoclonalantibody 266 and wherein the antibodies retain approximately the bindingproperties of the mouse antibody and have in vitro and in vivoproperties functionally equivalent to the mouse antibody (sequences SEQID NO:1 through SEQ ID NO:6). In another aspect, this invention provideshumanized antibodies and fragments thereof, wherein the variable regionshave sequences comprising the CDR from mouse antibody 266 and specifichuman framework sequences (sequences SEQ ID NO:7-SEQ ID NO:10), whereinthe antibodies retain approximately the binding properties of the mouseantibody and have in vitro and in vivo properties functionallyequivalent to the mouse antibody 266. In another aspect, this inventionprovides humanized antibodies and fragments thereof, wherein the lightchain is SEQ ID NO:11 and the heavy chain is SEQ ID NO:12.

Also part of the invention are polynucleotide sequences that encode thehumanized antibodies or fragments thereof disclosed above, vectorscomprising the polynucleotide sequences encoding the humanizedantibodies or fragments thereof, host cells transformed with the vectorsor incorporating the polynucleotides that express the humanizedantibodies or fragments thereof, pharmaceutical formulations of thehumanized antibodies and fragments thereof disclosed herein, and methodsof making and using the same.

Such humanized antibodies and fragments thereof are useful forsequestering Aβ in humans; for treating and preventing diseases andconditions characterized by Aβ plaques or Aβ toxicity in the brain, suchas Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathyin humans; for diagnosing these diseases in humans; and for determiningwhether a human subject will respond to treatment using human antibodiesagainst Aβ.

Administration of an appropriate humanized antibody in vivo to sequesterAβ peptide circulating in biological fluids is useful for preventive andtherapeutic treatment of conditions associated with the formation ofAβ-containing diffuse, neuritic, and cerebrovascular plaques in thebrain. The humanized antibody, including an immunologically reactivefragment thereof, results in removal of the Aβ peptide frommacromolecular complexes which would normally be relevant intransporting it in body fluids to and from sites where plaques can formor where it can be toxic. In addition, sequestering of plasma Aβ peptidewith the antibody or fragment thereof behaves as a “sink,” effectivelysequestering soluble Aβ peptide in the plasma compartment, and inducingAβ to enter the plasma from locations in the central nervous system(CNS). By sequestering Aβ in the blood, net efflux from the brain isenhanced and soluble Aβ is prevented from depositing in insolubleplaques and from forming toxic soluble species in the brain. Inaddition, insoluble Aβ in plaques which is in equilibrium with solubleAβ can be removed from the brain through a sequestering effect in theblood. Sequestering the Aβ peptide with the antibody also enhances itsremoval from the body and inhibits toxic effects of soluble Aβ in thebrain and the development and further accumulation of insoluble Aβ asamyloid in plaques. The antibodies useful in the invention do not crossthe blood-brain barrier in large amounts (≦0.1% plasma levels). Inaddition, humanized antibodies used in the invention, when administeredperipherally, do not need to elicit a cellular immune response in brainwhen bound to Aβ peptide or when freely circulating to have theirbeneficial effects. Further, when administered peripherally they do notneed to appreciably bind aggregated Aβ peptide in the brain to havetheir beneficial effects.

Thus, in one aspect, the invention is directed to a method to treat andto prevent conditions characterized by the formation of plaquescontaining beta-amyloid protein in humans, which method comprisesadministering, preferably peripherally, to a human in need of suchtreatment a therapeutically or prophylactically effective amount ofhumanized monoclonal antibody or immunologically reactive fragmentthereof, which antibody specifically binds to the mid-region of the Aβpeptide. In another aspect, the invention is directed to a method toinhibit the formation of amyloid plaques and to clear amyloid plaques inhumans, which method comprises administering to a human subject in needof such inhibition an effective amount of a humanized antibody thatsequesters Aβ peptide from its circulating form in blood and inducesefflux out of the brain as well as altered Aβ clearance in plasma andthe brain. In additional aspects, the invention is directed to suchhumanized antibodies, including immunologically effective portionsthereof, and to methods for their preparation.

The invention also includes methods of reversing cognitive decline,improving cognition, treating cognitive decline, and preventingcognitive decline in a subject diagnosed with clinical or pre-clinicalAlzheimer's disease, Down's syndrome, or clinical or pre-clinicalcerebral amyloid angiopathy, comprising administering to the subject aneffective amount of a humanized antibody of the invention.

The invention also includes use of a humanized antibody of the inventionfor the manufacture of a medicament, including prolonged expression ofrecombinant sequences of the antibody or antibody fragment in humantissues, for treating, preventing, or reversing Alzheimer's disease,Down's syndrome, or cerebral amyloid angiopathy; for treating,preventing, or reversing cognitive decline in clinical or pre-clinicalAlzheimer's disease, Down's syndrome, or clinical or pre-clinicalcerebral amyloid angiopathy; or to inhibit the formation of amyloidplaques or the effects of toxic soluble Aβ species in humans.

The invention is related to the surprising observation that within ashort period of time after administration of an antibody of the presentinvention, relatively large quantities of Aβ efflux from the centralnervous system to the blood. Thus, this invention includes methods toassess the response of a human subject to treatment with an antibodythat binds Aβ or a fragment thereof, comprising: a) administering theantibody or a fragment thereof to the subject; and b) measuring theconcentration of Aβ in the subject's blood.

The invention also includes a method of treating a human subject with anantibody that binds Aβ or a fragment thereof, comprising: a)administering a first amount of the antibody or fragment thereof to thesubject; b) within 3 hours to two weeks after administering the firstdose, measuring the concentration of Aβ in the subject's blood; c) ifnecessary, calculating a second amount of antibody or fragment thereofbased on the result of step b), which second amount is the same as ordifferent than the first amount; and d) administering the second amountof the antibody or fragment.

The invention also includes a method of assessing in a human subject theefficacy of an antibody that binds to Aβ, or a fragment thereof, forinhibiting or preventing Aβ amyloid plaque formation, for reducing Aβamyloid plaque, for reducing the effects of toxic soluble Aβ species, orfor treating a condition or a disease associated with Aβ plaque,comprising: a) obtaining a first sample of the subject's plasma or CSF;b) measuring a baseline concentration of Aβ in the first sample; c)administering the antibody or fragment thereof to the subject; d) within3 hours to two weeks after administering the antibody or fragmentthereof, obtaining a second sample of the subject's plasma or CSF; ande) measuring the concentration of Aβ in the second sample; wherein,efficacy is related to the quantity of Aβ bound to the antibody in theblood and the concentration of Aβ in the CSF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percentage of the Aβ peptide withdrawn from humancerebrospinal fluid through a dialysis membrane by Mab 266 as a functionof the molecular weight cutoff of the dialysis membrane.

FIG. 2 shows the concentration of Aβ_(Total) found in the plasma of anAPP^(V717F) transgenic mouse after injection with either 200 μg or 600μg of Mab 266 as a function of time.

FIG. 3A shows the quantity of Aβ peptide deposition in the cortex inAPP^(V717F) transgenic mice treated with saline, mouse IgG, or Mab 266.FIG. 3B shows correlation of these results with parental origin.

FIG. 4 shows the polynucleotide sequences for expressing 266 light chainfrom plasmid pVk-Hu266 and the single amino acid codes for the expressedhumanized 266 light chain (corresponding to SEQ ID NO:11 when mature).The complete sequence of the Hu266 light chain gene is located betweenthe MIuI and BamHI sites in pVk-Hu266. The nucleotide number indicatesits position in pVgk-Hu266. The V_(k) and C_(k) exons are translated insingle letter code; the dot indicates the translation termination codon.The mature heavy chain starts at the double-underlined aspartic acid(D). The intron sequences are in italic.

FIG. 5 shows the polynucleotide sequences for expressing 266 heavy chainfrom plasmid pVg1-Hu266 and the single amino acid codes for theexpressed humanized 266 heavy chain (corresponding to SEQ ID NO:12 whenmature). The complete sequence of the Hu266 heavy chain gene is locatedbetween the MIuI and BamHI sites in pVg1-Hu266. The nucleotide numberindicates its position in pVg1-Hu266. The V_(h) and C_(h) exons aretranslated in single letter code; the dot indicates the translationtermination codon. The mature heavy chain starts at thedouble-underlined glutamic acid (E). The intron sequences are in italic.

FIG. 6 is a plasmid map of pVk-Hu266.

FIG. 7 is a plasmid map of pVg1-Hu266.

MODES OF CARRYING OUT THE INVENTION

The Aβ peptides that circulate in human biological fluids represent thecarboxy terminal region of a precursor protein encoded on chromosome 21.It has been reported from the results of in vitro experiments that theAβ peptide has poor solubility in physiological solutions, since itcontains a stretch of hydrophobic amino acids which are a part of theregion that anchors its longer precursor to the lipid membranes ofcells. It is thus not surprising that circulating Aβ peptide is normallycomplexed with other moieties that prevent it from aggregating. This hasresulted in difficulties in detecting circulating Aβ peptide inbiological fluids.

The above-mentioned patent documents (U.S. Pat. Nos. 5,766,846;5,837,672 and 5,593,846) describe the preparation of antibodies,including a monoclonal antibody, designated clone 266 which was raisedagainst, and has been shown to bind specifically to, a peptidecomprising amino acids 13–28 of the Aβ peptide. The present applicantshave found that antibodies that bind within this region, in contrast toantibodies that bind elsewhere in the amino acid sequence of Aβ, areable to sequester the soluble Aβ peptide very effectively frommacromolecular complexes. This sequestration will effect net Aβ peptideefflux from the CNS, alter its clearance in CNS and plasma, and reduceits availability for plaque formation. Thus, antibodies of thisspecificity, modified to reduce their immunogenicity by converting themto a humanized form, offer the opportunity to treat, bothprophylactically and therapeutically, conditions that are associatedwith formation of beta-amyloid plaques. These conditions include, asnoted above, pre-clinical and clinical Alzheimer's, Down's syndrome, andpre-clinical and clinical cerebral amyloid angiopathy.

As used herein, the word “treat” includes therapeutic treatment, where acondition to be treated is already known to be present andprophylaxis—i.e., prevention of, or amelioration of, the possible futureonset of a condition.

By “monoclonal antibodies that bind to the mid-region of Aβ peptide” ismeant monoclonal antibodies (Mab or Mabs) that bind an amino acidsequence representing an epitope contained between positions 13–28 ofAβ. The entire region need not be targeted. As long as the antibodybinds at least an epitope within this region (especially, e.g.,including the α-secretase site 16–17 or the site at which antibody 266binds), such antibodies are effective in the method of the invention.

By “antibody” is meant a monoclonal antibody per se, or animmunologically effective fragment thereof, such as an F_(ab), F_(ab′),or F_((ab′)2) fragment thereof. In some contexts, herein, fragments willbe mentioned specifically for emphasis; nevertheless, it will beunderstood that regardless of whether fragments are specified, the term“antibody” Includes such fragments as well as single-chain forms. Aslong as the protein retains the ability specifically to bind itsintended target, and in this case, to sequester Aβ peptide from itscarrier proteins in blood, it is included within the term “antibody.”Also included within the definition “antibody” for example, are singlechain forms, generally designated F_(v) regions, of antibodies with thisspecificity. Preferably, but not necessarily, the antibodies useful inthe invention are produced recombinantly, as manipulation of thetypically murine or other non-human antibodies with the appropriatespecificity is required in order to convert them to humanized form.Antibodies may or may not be glycosylated, though glycosylatedantibodies are preferred. Antibodies are properly cross-linked viadisulfide bonds, as is well-known.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50–70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as gamma, mu, alpha, and lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, and definethe antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids.

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. The chainsall exhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarily determining regions or CDRs. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith well known conventions [Kabat “Sequences of Proteins ofImmunological Interest” National Institutes of Health, Bethesda, Md.,1987 and 1991; Chothia, et al., J. Mol. Biol. 196:901–917 (1987);Chothia, et al., Nature 342:878–883 (1989)].

As is well understood in the art, monoclonal antibodies can readily begenerated with appropriate specificity by standard techniques ofimmunization of mammals, forming hybridomas from the antibody-producingcells of said mammals or otherwise immortalizing them, and culturing thehybridomas or immortalized cells to assess them for the appropriatespecificity. In the present case such antibodies could be generated byimmunizing a human, rabbit, rat or mouse, for example, with a peptiderepresenting an epitope encompassing the 13–28 region of the Aβ peptideor an appropriate subregion thereof. Materials for recombinantmanipulation can be obtained by retrieving the nucleotide sequencesencoding the desired antibody from the hybridoma or other cell thatproduces it. These nucleotide sequences can then be manipulated toprovide them in humanized form.

By “humanized antibody” is meant an antibody that is composed partiallyor fully of amino acid sequences derived from a human antibody germlineby altering the sequence of an antibody having non-human complementaritydetermining regions (CDR). The simplest such alteration may consistsimply of substituting the constant region of a human antibody for themurine constant region, thus resulting in a human/murine chimera whichmay have sufficiently low immunogenicity to be acceptable forpharmaceutical use. Preferably, however, the variable region of theantibody and even the CDR is also humanized by techniques that are bynow well known in the art. The framework regions of the variable regionsare substituted by the corresponding human framework regions leaving thenon-human CDR substantially intact, or even replacing the CDR withsequences derived from a human genome. Fully human antibodies areproduced in genetically modified mice whose immune systems have beenaltered to correspond to human immune systems. As mentioned above, it issufficient for use in the methods of the invention, to employ animmunologically specific fragment of the antibody, including fragmentsrepresenting single chain forms.

A humanized antibody again refers to an antibody comprising a humanframework, at least one CDR from a non-human antibody, and in which anyconstant region present is substantially identical to a humanimmunoglobulin constant region, i.e., at least about 85–90%, preferablyat least 95% identical. Hence, all parts of a humanized antibody, exceptpossibly the CDRs, are substantially identical to corresponding parts ofone or more native human immunoglobulin sequences. For example, ahumanized immunoglobulin would typically not encompass a chimeric mousevariable region/human constant region antibody.

Humanized antibodies have at least three potential advantages overnon-human and chimeric antibodies for use in human therapy:

1) because the effector portion is human, it may interact better withthe other parts of the human immune system (e.g., destroy the targetcells more efficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC)).

2) The human immune system should not recognize the framework or Cregion of the humanized antibody as foreign, and therefore the antibodyresponse against such an injected antibody should be less than against atotally foreign non-human antibody or a partially foreign chimericantibody.

3) Injected non-human antibodies have been reported to have a half-lifein the human circulation much shorter than the half-life of humanantibodies. Injected humanized antibodies will have a half-lifeessentially identical to naturally occurring human antibodies, allowingsmaller and less frequent doses to be given.

The design of humanized immunoglobulins may be carried out as follows.When an amino acid falls under the following category, the frameworkamino acid of a human immunoglobulin to be used (acceptorimmunoglobulin) is replaced by a framework amino acid from aCDR-providing non-human immunoglobulin (donor immunoglobulin):

(a) the amino acid in the human framework region of the acceptorimmunoglobulin is unusual for human immunoglobulin at that position,whereas the corresponding amino acid in the donor immunoglobulin istypical for human immunoglobulin at that position;

(b) the position of the amino acid is immediately adjacent to one of theCDRs; or

(c) any side chain atom of a framework amino acid is within about 5–6angstroms (center-to-center) of any atom of a CDR amino acid in a threedimensional immunoglobulin model [Queen, et al., op. cit., and Co, etal., Proc. Natl. Acad. Sci. USA 88, 2869 (1991)]. When each of the aminoacid in the human framework region of the acceptor immunoglobulin and acorresponding amino acid in the donor immunoglobulin is unusual forhuman immunoglobulin at that position, such an amino acid is replaced byan amino acid typical for human immunoglobulin at that position.

A preferred humanized antibody is a humanized form of mouse antibody266. The CDRs of humanized 266 have the following amino acid sequences:

light chain CDR1: 1               5                   10                  15 Arg Ser SerGln Ser Leu Ile Tyr Ser Asp Gly Asn Ala Tyr Leu His (SEQ ID NO:1) lightchain CDR2:  1               5 Lys Val Ser Asn Arg Phe Ser (SEQ ID NO:2)light chain CDR3: 1                5 Ser Gln Ser Thr His Val Pro Trp Thr(SEQ ID NO:3) heavy chain CDR1:  1               5 Arg Tyr Ser Met Ser(SEQ ID NO:4) heavy chain CDR2: 1               5                   10                  15 Gln Ile AsnSer Val Gly Asn Ser Thr Tyr Tyr Pro Asp Thr Val Lys Gly (SEQ ID NO:5)and, heavy chain CDR3:  1 Gly Asp Tyr. (SEQ ID NO:6)A preferred light chain variable region of a humanized antibody of thepresent invention has the following amino acid sequence, in which theframework originated from human germline Vk segments DPK18 and J segmentJk1, with several amino acid substitutions to the consensus amino acidsin the same human V subgroup to reduce potential immunogenicity:

1                5                   10                   15 Asp Xaa ValMet Thr Gln Xaa Pro Leu Ser Leu Pro Val Xaa Xaa (SEQ ID NO:7)                 20                  25                   30 Gly Gln ProAla Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Xaa                 35                  40                   45 Tyr Ser AspGly Asn Ala Tyr Leu His Trp Phe Leu Gln Lys Pro                 50                  55                   60 Gly Gln SerPro Xaa Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe                 65                  70                   75 Ser Gly ValPro Asp Ary Phe Ser Gly Ser Gly Ser Gly Thr Asp                 80                  85                   90 Phe Thr LeuLys Ile Ser Arg Val Glu Ala Glu Asp Xaa Gly Val                 95                  100                 105 Tyr Tyr CysSer Gln Ser Thr His Val Pro Trp Thr Phe Gly Xaa                 110 GlyThr Xaa Xaa Glu Ile Lys Arg

wherein:

Xaa at position 2 is Val or Ile;

Xaa at position 7 is Ser or Thr;

Xaa at position 14 is Thr or Ser;

Xaa at position 15 is Leu or Pro;

Xaa at position 30 is Ile or Val;

Xaa at position 50 is Arg, Gln, or Lys;

Xaa at position 88 is Val or Leu;

Xaa at position 105 is Gin or Gly;

Xaa at position 108 is Lys or Arg; and

Xaa at position 109 is Val or Leu.

A preferred heavy chain variable region of a humanized antibody of thepresent invention has the following amino acid sequence, in which theframework originated from human germline VH segments DP53 and J segmentJH4, with several amino acid substitutions to the consensus amino acidsin the same human subgroup to reduce potential immunogenicity:

 1               5                   10                   15 Xaa Val GlnLeu Val Glu Xaa Gly Gly Gly Leu Val Gln Pro Gly (SEQ ID NO:8)                 20                  25                   30 Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser                 35                  40                   45 Arg Tyr SerMet Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                 50                  55                   60 Xaa Leu ValAla Gln Ile Asn Ser Val Gly Asn Ser Thr Tyr Tyr                 65                  70                   75 Pro Asp XaaVal Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa                 80                  85                   90 Xaa Asn ThrLeu Tyr Leu Gln Met Asn Ser Leu Arg Ala Xaa Asp                 95                  100                 105 Thr Ala ValTyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly                 110 ThrXaa Val Thr Val Ser Ser

wherein:

Xaa at position 1 is Glu or Gin;

Xaa at position 7 is Ser or Leu;

Xaa at position 46 is Glu, Val, Asp, or Ser;

Xaa at position 63 is Thr or Ser;

Xaa at position 75 is Ala, Ser, Val, or Thr;

Xaa at position 76 is Lys or Arg;

Xaa at position 89 is Glu or Asp; and

Xaa at position 107 is Leu or Thr.

A particularly preferred light chain variable region of a humanizedantibody of the present invention has the following amino acid sequence,in which the framework originated from human germline Vk segments DPK18and J segment Jk1, with several amino acid substitutions to theconsensus amino acids in the same human V subgroup to reduce potentialimmunogenicity:

1                5                   10                   15 Asp Val ValMet Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu (SEQ ID NO:9)                 20                  25                   30 Gly Gln ProAla Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile                 35                  40                   45 Tyr Ser AspGly Asn Ala Tyr Leu His Trp Phe Len Gln Lys Pro                 50                  55                   60 Gly Gln SerPro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe                 65                  70                   75 Ser Gly ValPro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp                 80                  85                   90 Phe Thr LeuLys Ile Ser Arg Val Glu Ala Gln Asp Val Gly Val                 95                  100                 105 Tyr Tyr CysSer Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln                 110 GlyThr Lys Val Gln Ile Lys Arg.

A particularly preferred heavy chain variable region of a humanizedantibody of the present invention has the following amino acid sequence,in which the framework originated from human germline VH segments DP53and J segment JH4:

 1               5                   10                   15 Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly (SEQ ID NO:10)                 20                  25                   30 Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser                 35                  40                   45 Arg Tyr SerMet Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                 50                  55                   60 Glu Leu ValAla Gln Ile Asn Ser Val Gly Asn Ser Thr Tyr Tyr                 65                  70                   75 Pro Asp ThrVal Lys Gly Ary Phe Thr Ile Ser Arg Asp Asn Ala                 80                  85                   90 Lys Asn ThrLeu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                 95                  100                 105 Thr Ala ValTyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly                 110 ThrLeu Val Thr Val Ser Ser.

A preferred light chain for a humanized antibody of the presentinvention has the amino acid sequence:

 1               5                   10                   15 Asp Val ValMet Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu (SEQ ID NO:11)                 20                  25                   30 Gly Gln ProAla Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile                 35                  40                   45 Tyr Ser AspGly Asn Ala Tyr Leu His Trp Phe Leu Gln Lys Pro                 50                  55                   60 Gly Gln SerPro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe                 65                  70                   75 Ser Gly ValPro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp                 80                  85                   90 Phe Thr LeuLys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val                 95                  100                 105 Tyr Tyr CysSer Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln                110                  115                 120 Gly Thr LysVal Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val                125                  130                 135 Phe Ile PhePro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala                140                  145                 150 Ser Val ValCys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys                155                  160                 165 Val Gln TrpLys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln                170                  175                 180 Glu Ser ValThr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                185                  190                 195 Ser Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys                200                  205                 210 Val Tyr AlaCys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val                 215 ThrLys Ser Phe Asn Arg Gly Glu Cys.

A preferred heavy chain for a humanized antibody of the presentinvention has the amino acid sequence:

  1              5                   10                   15 Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly (SEQ ID NO:12)                 20                  25                   30 Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser                 35                  40                   45 Arg Tyr SerMet Ser Trp Val Ary Gln Ala Pro Gly Lys Gly Leu                 50                  55                   60 Glu Leu ValAla Gln Ile Asn Ser Val Gly Asn Ser Thr Tyr Tyr                 65                  70                   75 Pro Asp ThrVal Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala                 80                  85                   90 Lys Asn ThrLeu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                 95                  100                 105 Thr Ala ValTyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly                110                  115                 120 Thr Leu ValThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val                125                  130                 135 Phe Pro LeuAla Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala                140                  145                 150 Ala Leu GlyCys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr                155                  160                 165 Val Ser TrpAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                170                  175                 180 Pro Ala ValLeu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val                185                  190                 195 Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys                200                  205                 210 Asn Val AsnHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val                215                  220                 225 Glu Pro LysSer Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro                230                  235                 240 Ala Pro GluLeu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro                245                  250                 255 Lys Pro LysAsp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr                260                  265                 270 Cys Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys Phe                275                  280                 285 Asn Trp TyrVal Asp Gly Val Glu Val His Asn Ala Lys Thr Lys                290                  295                 300 Pro Arg GluGlu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val                305                  310                 315 Leu Thr ValLeu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys                320                  325                 330 Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr                335                  340                 345 Ile Ser LysAla Lys Gly Gln Pro Ary Glu Pro Gln Val Tyr Thr                350                  355                 360 Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu                365                  370                 375 Thr Cys LeuVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu                380                  385                 390 Trp Glu SerAsn Gly Gln Pro Glu Aso Asn Tyr Lys Thr Thr Pro                395                  400                 405 Pro Val LeuAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu                410                  415                 420 Thr Val AspLys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys                425                  430                 435 Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser                 440 LeuSer Leu Ser Pro Gly Lys.

Other sequences are possible for the light and heavy chains for thehumanized antibodies of the present invention and for humanized 266. Theimmunoglobulins can have two pairs of light chain/heavy chain complexes,at least one chain comprising one or more mouse complementaritydetermining regions functionally joined to human framework regionsegments.

In another aspect, the present invention is directed to recombinantpolynucleotides encoding antibodies which, when expressed, comprise theheavy and light chain CDRs from an antibody of the present invention. Asto the human framework region, a framework or variable region amino acidsequence of a CDR-providing non-human immunoglobulin is compared withcorresponding sequences in a human immunoglobulin variable regionsequence collection, and a sequence having a high percentage ofidentical amino acids is selected. Exemplary polynucleotides, which onexpression code for the polypeptide chains comprising the heavy andlight chain CDRs of monoclonal antibody 266 are given in FIGS. 4 and 5.Due to codon degeneracy and non-critical amino-acid substitutions, otherpolynucleotide sequences can be readily substituted for those sequences.Particularly preferred polynucleotides of the present invention encodeantibodies, which when expressed, comprise the CDRs of SEQ ID NO:1–SEQID NO:6, or any of the variable regions of SEQ ID NO:7–SEQ ID NO:10, orthe light and heavy chains of SEQ ID NO:11 and SEQ ID NO:12.

The polynucleotides will typically further include an expression controlpolynucleotide sequence operably linked to the humanized immunoglobulinencoding sequences, including naturally-associated or heterologouspromoter regions. Preferably, the expression control sequences will beeukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells, but control sequences forprokaryotic hosts may also be used. Once the vector has beenincorporated into the appropriate host cell line, the host cell ispropagated under conditions suitable for high level expression of thenucleotide sequences, and, as desired, the collection and purificationof the light chains, heavy chains, light/heavy chain dimers or intactantibodies, binding fragments or other immunoglobulin forms may follow.

The nucleotide sequences of the present invention capable of ultimatelyexpressing the desired humanized antibodies can be formed from a varietyof different polynucleotides (genomic or cDNA, RNA, syntheticoligonucleotides, etc.) and components (e.g., V, J, D, and C regions),as well as by a variety of different techniques. Joining appropriategenomic and synthetic sequences is a common method of production, butcDNA sequences may also be utilized.

Human constant region DNA sequences can be isolated in accordance withwell known procedures from a variety of human cells, but preferably fromimmortalized B-cells. The CDRs for producing the immunoglobulins of thepresent invention will be similarly derived from non-human monoclonalantibodies capable of binding to an epitope between amino acids 13 and28 of the Aβ peptide, which monoclonal antibodies are produced in anyconvenient mammalian source, including, mice, rats, rabbits, or othervertebrates capable of producing antibodies by well known methods, asdescribed above. Suitable source cells for the polynucleotide sequencesand host cells for immunoglobulin expression and secretion can beobtained from a number of sources well-known in the art.

In addition to the humanized immunoglobulins specifically describedherein, other “substantially homologous” modified immunoglobulins can bereadily designed and manufactured utilizing various recombinant DNAtechniques well known to those skilled in the art. For example, theframework regions can vary from the native sequences at the primarystructure level by several amino acid substitutions, terminal andintermediate additions and deletions, and the like. Moreover, a varietyof different human framework regions may be used singly or incombination as a basis for the humanized immunoglobulins of the presentinvention. In general, modifications of the genes may be readilyaccomplished by a variety of well-known techniques, such assite-directed mutagenesis.

Alternatively, polypeptide fragments comprising only a portion of theprimary antibody structure may be produced, which fragments possess oneor more immunoglobulin activities (e.g., complement fixation activity).These polypeptide fragments may be produced by proteolytic cleavage ofintact antibodies by methods well known in the art, or by inserting stopcodons at the desired locations in vectors using site-directedmutagenesis, such as after CH1 to produce Fab fragments or after thehinge region to produce F(ab′)₂ fragments. Single chain antibodies maybe produced by joining VL and VH with a DNA linker.

As stated previously, the encoding nucleotide sequences will beexpressed in hosts after the sequences have been operably linked to(i.e., positioned to ensure the functioning of) an expression controlsequence. These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors will contain selectionmarkers, e.g., tetracycline or neomycin, to permit detection of thosecells transformed with the desired DNA sequences.

E. coli is a prokaryotic host useful particularly for cloning thepolynucleotides of the present invention. Other microbial hosts suitablefor use include bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any of a number of well-known promoters maybe present, such as the lactose promoter system, a tryptophan (trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters will typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences and the like, for initiating and completing transcription andtranslation.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention.Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting intact immunoglobulins have beendeveloped in the art, and include the CHO cell lines, various COS celllines, Syrian Hamster Ovary cell lines, HeLa cells, preferably myelomacell lines, transformed B-cells, human embryonic kidney cell lines, orhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, cytomegalovirus and the like.

The vectors containing the nucleotide sequences of interest (e.g., theheavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment or electroporation may be used forother cellular hosts.

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe purified according to standard procedures of the art, includingammonium sulfate precipitation, ion exchange, affinity, reverse phase,hydrophobic interaction column chromatography, gel electrophoresis andthe like. Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically or prophylactically, as directed herein.

The antibodies (including immunologically reactive fragments) areadministered to a subject at risk for or exhibiting Aβ-related symptomsor pathology such as clinical or pre-clinical Alzheimer's disease,Down's syndrome, or clinical or pre-clinical amyloid angiopathy, usingstandard administration techniques, preferably peripherally (i.e. not byadministration into the central nervous system) by intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. Althoughthe antibodies may be administered directly into the ventricular system,spinal fluid, or brain parenchyma, and techniques for addressing theselocations are well known in the art, it is not necessary to utilizethese more difficult procedures. The antibodies of the invention areeffective when administered by the more simple techniques that rely onthe peripheral circulation system. The advantages of the presentinvention include the ability of the antibody exert its beneficialeffects even though not provided directly to the central nervous systemitself. Indeed, it has been demonstrated herein that the amount ofantibody which crosses the blood-brain barrier is <0.1% of plasma levelsand that the antibodies of the invention exert their ability tosequester Aβ in the peripheral circulation as well as to alter CNS andplasma soluble Aβ clearance.

The pharmaceutical compositions for administration are designed to beappropriate for the selected mode of administration, andpharmaceutically acceptable excipients such as dispersing agents,buffers, surfactants, preservatives, solubilizing agents, isotonicityagents, stabilizing agents and the like are used as appropriate.Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa.,latest edition, incorporated herein by reference, provides a compendiumof formulation techniques as are generally known to practitioners. Itmay be particularly useful to alter the solubility characteristics ofthe antibodies of the invention, making them more lipophilic, forexample, by encapsulating them in liposomes or by blocking polar groups.

Peripheral systemic delivery by intravenous or intraperitoneal orsubcutaneous injection is preferred. Suitable vehicles for suchinjections are straightforward. In addition, however, administration mayalso be effected through the mucosal membranes by means of nasalaerosols or suppositories. Suitable formulations for such modes ofadministration are well known and typically include surfactants thatfacilitate cross-membrane transfer. Such surfactants are often derivedfrom steroids or are cationic lipids, such asN-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammoniumchloride (DOTMA) orvarious compounds such as cholesterol hemisuccinate, phosphatidylglycerols and the like.

The concentration of the humanized antibody in formulations from as lowas about 0.1% to as much as 15 or 20% by weight and will be selectedprimarily based on fluid volumes, viscosities, and so forth, inaccordance with the particular mode of administration selected. Thus, atypical pharmaceutical composition for injection could be made up tocontain 1 mL sterile buffered water of phosphate buffered saline and1–100 mg of the humanized antibody of the present invention. Theformulation could be sterile filtered after making the formulation, orotherwise made microbiologically acceptable. A typical composition forintravenous infusion could have a volume as much as 250 mL of fluid,such as sterile Ringer's solution, and 1–100 mg per mL, or more inantibody concentration. Therapeutic agents of the invention can befrozen or lyophilized for storage and reconstituted in a suitablesterile carrier prior to use. Lyophilization and reconstitution can leadto varying degrees of antibody activity loss (e.g. with conventionalimmune globulins, IgM antibodies tend to have greater activity loss thanIgG antibodies). Dosages may have to be adjusted to compensate. The pHof the formulation will be selected to balance antibody stability(chemical and physical) and comfort to the patient when administered.Generally, pH between 4 and 8 is tolerated.

Although the foregoing methods appear the most convenient and mostappropriate for administration of proteins such as humanized antibodies,by suitable adaptation, other techniques for administration, such astransdermal administration and oral administration may be employedprovided proper formulation is designed.

In addition, it may be desirable to employ controlled releaseformulations using biodegradable films and matrices, or osmoticmini-pumps, or delivery systems based on dextran beads, alginate, orcollagen.

In summary, formulations are available for administering the antibodiesof the invention and are well-known in the art and may be chosen from avariety of options.

Typical dosage levels can be optimized using standard clinicaltechniques and will be dependent on the mode of administration and thecondition of the patient.

The following examples are intended to illustrate but not to limit theinvention.

The examples hereinbelow employ, among others, a murine monoclonalantibody designated “266” which was originally prepared by immunizationwith a peptide composed of residues 13–28 of human Aβ peptide. Theantibody was confirmed to immunoreact with this peptide, but hadpreviously been reported to not react with the peptide containing onlyresidues 17–28 of human Aβ peptide, or at any other epitopes within theAβ peptide. The preparation of this antibody is described in U.S. Pat.No. 5,766,846, incorporated herein by reference. As the examples heredescribe experiments conducted in murine systems, the use of murinemonoclonal antibodies is satisfactory. However, in the treatment methodsof the invention intended for human use, humanized forms of theantibodies with the immunospecificity corresponding to that of antibody266 are preferred.

EXAMPLE 1 Sequestration of Added Aβ Peptide in Human Fluids

Samples of human cerebrospinal fluid (CSF) (50 μl) and human plasma (50μl) were incubated for 1 hour at room temperature as follows:

1. alone;

2. along with 5 ng Aβ 40 peptide; or

3.5 ng Aβ 40 peptide plus 1 mg monoclonal antibody 266 (described, forexample, in U.S. Pat. No. 5,766,846 incorporated herein by reference).

The samples were then electrophoresed on a 4–25% non-denaturing gradientgel, i.e., non-denaturing gradient electrophoresis (NDGGE) andtransferred to nitrocellulose. The blots were then stained with PonceauS or, for Western blot, probed with biotin-labeled monoclonal antibody(3D6) which is directed against the first five amino acids of Aβpeptide, developed with streptavidin-horse radish peroxidase anddetected by enhanced chemiluminescence (ECL). The hydrated diameters ofthe materials contained in bands on the blots were estimated usingPharmacia molecular weight markers. Thus, if the Aβ peptide is bound toother molecules, it would run at the size of the resulting complex.

Western blots of CSF either with or without 5 ng Aβ peptide shows noevidence of the Aβ peptide in response to detection mediated by antibody3D6. Similar results are obtained for human plasma. This was truedespite the fact that Aβ peptide could be detected by SDS-PAGE followedby Western blot using the same technique and on the same CSF samples.Presumably, the detection of Aβ peptide was prevented by interactionsbetween this peptide and other factors in the fluids tested. However,when Mab 266 is added to the incubation, characteristic bandsrepresenting sequestered Aβ peptide complexed to the antibody arepresent both in plasma and in CSF. The major band is at approximately 11nm hydrated diameter, corresponding to antibody monomer with anadditional smaller band at 13 nm corresponding to antibody dimer.

EXAMPLE 2 Specificity of the Sequestering Antibody

Samples containing 50 μl of human CSF or 10 μl of APP^(V717F) CSF wereused. APP^(V717F) are transgenic mice representing a mouse model ofAlzheimer's disease in which the human amyloid precursor proteintransgene with a familial Alzheimer's disease mutation is expressed andresults in the production of human Aβ peptide in the central nervoussystem.

The samples were incubated with or without various Mabs (1 μg) for 1hour at room temperature and then electrophoresed on a 4–25% NDGGE andblotted onto nitrocellulose as described in Example 1. The antibodieswere as follows:

Mab 266 (binds to positions 13–28);

Mab 4G8 (binds to positions 17–24);

QCBpan (rabbit polyclonal for positions 1–40);

mouse IgG (non-specific);

Mab 3D6 (binds to positions 1–5);

Mab 21F12 (binds to positions 33–42):

Mab 6E10 (binds to positions 1–17); and

QCB_(40,42) (rabbit polyclonals to Aβ₄₀ and Aβ₄₂).

Detection of the Aβ peptide antibody complex was as described in Example1—biotin labeled 3D6 (to the Aβ peptide N-terminus) followed bystreptavidin-HRP and ECL. Similar detection in human CSF incubated withMab 266, in some instances substituted QCB_(40,42), which binds to thecarboxyl terminus of Aβ peptide, for 3D6.

The results showed that of the antibodies tested, only Mab 4G8 and Mab266 permitted the detection of Aβ peptide.

The results showed that for human CSF, only Mab 266 and Mab 4G8 wereable to sequester in detectable amounts of an antibody Aβ complex(again, without any antibody, no Aβ is detected). Mab 266 was also ableto produce similar results to those obtained with human CSF with CSFfrom APP^(V717F) transgenic mice. Aβ peptide could be sequestered inhuman CSF using Mab 266 regardless of whether 3D6 or QCB_(40,42)antibody was used to develop the Western blot.

EXAMPLE 3 Demonstration of Aβ3 Peptide-266 Complex by Two-DimensionalElectrophoresis

A sample containing 50 ng Aβ₄₀ peptide was incubated with 2 μg Mab 266at 37° C. for 3 hours. A corresponding incubation of Mab 266 alone wasused as a control.

The samples were then subjected to 2-dimensional gel electrophoresis.

In the first dimension, the incubated samples were subjected to NDGGE asdescribed in Example 1. The polyacrylamide gel was then cut intoindividual lanes perpendicular to the direction of the first dimensionalflow and gel separation under denaturing/reducing conditions by SDS-PAGE(Tricine urea gel) was performed in the second dimension. The presenceof the bands was detected either by Ponceau-S staining (any protein) orby specific development using 6E10 Mab (Senetek, Inc.) and biotinylatedanti-mouse Aβ in the HRP-based detection system.

Ponceau-S staining of the nitrocellulose blots after transfer permittedvisualization of the heavy and light chains of Mab 266 alone. It wasconfirmed that Aβ peptide was in a complex with Mab 266 as a band at 4kD was observed that aligns with the size of full-length Mab 266 seenafter the first dimension NDGGE.

EXAMPLE 4 Demonstration of Non-Equivalence of Binding and Sequestration

Aβ peptide as it circulates in plasma and CSF is thought to be containedin a complex with proteins, including apolipoprotein E. The presentexample demonstrates that antibodies to apoE, while able to bind to thecomplex, do not sequester apoE from the remainder of the complex.

ApoE complexes (500 ng) were incubated with Mab or polyclonal antibodiesto apoE (2 μg) at 37° C. for one hour. The incubated samples were thensubjected to NDGGE using the techniques described in Example 1.Following NDGGE, Western blotting was performed with affinity purifiedgoat anti-apoE antibodies with detection by ECL. When no antibody ispresent, apoE can be detected at 8–13 nm consistent with its presence inlipoprotein particles. The presence of monoclonal or polyclonalantibodies to apoE results in a population shift of apoE to a largermolecular species, a “super shift”. This demonstrates that theantibodies to apoE did not sequester, i.e., remove apoE from alipoprotein particle, rather they bind to apoE on the lipoproteinscreating a larger molecular species.

EXAMPLE 5 Sequestration of Aβ is Not Perturbed by Anti-apoE Antibodies

A sample of 100 μl human CSF was incubated either with Mab 266 alone, orwith polyclonal anti-apoE, or with both antibodies for 60 minutes at 37°C. The samples were then analyzed by NDGGE as described in Example 1 andthe detection of bands performed as described in Example 1.

The results show that as long as Mab 266 was added to the sample, theband at approximately 11 nm diameter characteristic of the sequestered266-Aβ peptide complex was visible. This is the case whether or notanti-apoE is present. This band, demonstrating sequestered Aβ, alsoappears if 50 ng of Aβ peptide is added to the incubation mixture in thepresence of Mab 266. Thus, alteration of the molecular weight of apoE bythe presence of anti-apoE antibodies does not interfere withsequestration of Aβ peptide by Mab 266.

EXAMPLE 6 Sequestration of Aβ Peptide In Vivo

A. Transgenic APP^(V717F) mice, also termed PDAPP mice, over-express amutant form of human APP protein. These mice produce human Aβ in the CNSand have elevated levels of human Aβ peptide circulating in the CSF andplasma. Eight month old mice were injected intravenously with saline or100 μg of Mab 266. They were bled 10 minutes after initial injection andagain at 20 hours after initial injection.

Samples containing 20 μl of plasma from each animal were analyzed byNDGGE and Western blot with antibody 3D6 as described in Example 1. Thesaline injected animals did not show the presence of the characteristic11 nm sequestered Aβ peptide band either after 10 minutes or 20 hours.However, the two animals that were injected with Mab 266 did show theappearance of this band after 20 hours.

B. Two month old APP^(V717F) mice were used in this study. At day zero,the mice received either no Mab 266, 1 mg Mab 266, or 100 μg of thisantibody. Plasma samples were taken two days prior to administration ofthe antibodies and on days 1, 3, 5 and 7. The plasma samples weresubjected to NDGGE followed by Western blotting and detection with 3D6as described in Example 1. At all time points following administrationof Mab 266, the 266/Aβ complex was detected unless the plasma sample hadbeen treated with protein G, which binds to immunoglobulin, thuseffectively removing the Mab 266. Consistent levels of complex over thetime period tested were found except for a slight drop-off at day sevenin animals injected with 100 μg of Mab 266; in general, the levels inanimals administered 100 μg were consistently lower than those found inthe mice administered 1 mg of this antibody.

C. Two two-month old APP^(V717F) mice were administered 1 mg of Mab 266intravenously and a 25 μl plasma sample was taken from each. The plasmasample was subjected to NDGGE followed by Western blot as describedabove except that binding with biotinylated 3D6 was followed bydetection with streptavidin¹²⁵I (Amersham) and exposure to aphosphorimaging screen. The level of complex was estimated in comparisonto a standard curve using known amounts of Aβ₄₀ complexed withsaturating levels of Mab 266 and detected similarly. The amount of Aβpeptide bound to Mab 266 was estimated at approximately 100 ng/ml,representing an increase of approximately 1,000-fold over endogenous Aβpeptide in these mice which had been determined to be about 100 pg/ml.This is also similar to the level of Aβ peptide in APP^(V717F) brainprior to Aβ deposition (50–100 ng/g); human AβP and human Aβ inAPP^(V717F) Tg mice are produced almost solely in the brain. Thus, itappears that the presence of Mab 266 in the plasma acts as an Aβ peptidesink facilitating net efflux of Aβ peptide from the CNS into the plasma.This increased net efflux likely results from both increasing Aβ effluxfrom CNS to plasma and also from preventing Aβ in plasma fromre-entering the brain.

The correct size for the sequestered Aβ peptide was confirmed by running20 μL of plasma samples obtained from APP^(V717F) mice 24 hours afterbeing injected with 1 mg Mab 266 on TRIS-tricine SDS-PAGE gels followedby Western blotting using anti-Aβ antibody 6E10 prior to, or after,protein G exposure using protein G-bound beads. A band that was depletedby protein G was detected at 4–8 kD, consistent with the presence ofmonomers and possibly dimers of Aβ peptide.

D. Two month old APP^(V717F) mice were treated with either PBS (n=7) or500 μg biotinylated Mab 266—i.e., m266B (n=9) intraperitoneally. Bothprior to and 24 hours after the injection, plasma was analyzed for totalAβ peptide using a modification of the ELISA method of Johnson-Wood, K.,et al., Proc. Natl. Acad. Sci. USA (1997). 94:1550–1555; and Bales, K.R., et al., Nature Genet (1997) 17:263–264. Total Aβ bound to m266B wasmeasured by using 96-well Optiplates (Packard, Inc.) coated with m3D6.Diluted plasma samples and standards (varying concentrations of Aβ₄₀ andm266B) were incubated overnight in the coated plates and the amount oftotal Aβ/m266B complex was determined with the use of ¹²⁵I-Streptavidin.In addition, at the 24-hour time point, the plasma samples were firsttreated with protein G to quantitate Aβ peptide not bound to Mab 266,and Aβ_(Total) and Aβ₄₂ were determined by ELISA in the CSF. InPBS-injected animals, plasma Aβ peptide levels were 140 pg/ml bothbefore and after injection. Plasma levels were similar in the Mab266-injected mice prior to injection, but levels of Aβ peptide not boundto Mab 266 were undetectable at 24 hours post injection.

Levels in the CSF were also measured, CSF represents an extracellularcompartment within the CNS and concentration of molecules in the CSFreflects to some extent the concentration of substances in theextracellular space of the brain. CSF was isolated from the cisternamagna compartment. Mice were anesthetized with pentobarbital and themusculature from the base of the skull to the first vertebrae wasremoved. CSF was collected by carefully puncturing the arachnoidmembrane covering the cistern with a micro needle under a dissectingmicroscope and withdrawing the CSF into a polypropylene micropipette. At24 hours post injection, an increase in total Aβ peptide in the CSF ofMab 266-injected mice was found, and an approximately two-fold increasein Aβ₄₂ as compared to PBS injected mice was obtained in the CSF. Thiswas confirmed using denaturing gel electrophoresis followed by Westernblotting with Aβ₄₂-specific antibody 21F12.

In an additional experiment, three month old APP^(V717F) Tg mice wereinjected with either PBS or Mab 266 intravenously and both Aβ₄₀ and Aβ₄₂levels were assessed in the CSF as follows:

For measurement of Aβ₄₀, the monoclonal antibody m2G3, specific for Aβ₄₀was utilized. The ELISA described (Johnson-Wood, K., et al., Proc. Natl.Acad. Sci. USA (1997) 94:1550–1555) was modified into an RIA byreplacing the Streptavidin-HRP reagent with ¹²⁵I-Streptavidin. Forplasma and CSF samples, the procedure was performed under non-denaturingconditions that lacked guanidine in the buffers. For assessment ofcarbonate soluble and insoluble Aβ in brain homogenate, samples werehomogenized with 100 mM carbonate, 40 mM NaCl, pH 11.5 (4° C.), spun at10,000×g for 15 min, and Aβ was assessed in the supernatant (soluble)and the pellet (insoluble) fractions as described (Johnson-Wood, K., etal., Proc. Natl. Acad. Sci. USA (1997) 94:1550–1555) and listed above.The measurement of Aβ/Mab 266 complex in plasma was performed by amodified RIA. Mice were injected with biotinylated Mab 266 (Mab 266B)and plasma was isolated at multiple time points. Total Aβ bound to Mab266 was measured by using 96-well Optiplates (Packard, Inc.) coated withm3D6. Diluted plasma samples and standards (varying concentrations ofAβ₄₀ and Mab 266B) were incubated overnight in the coated plates and theamount of total Aβ/Mab 266B complex was determined with the use of¹²⁵¹I-Streptavidin.

Three hours following the intravenous injection of Mab 266, there was atwo-fold increase in CSF Aβ₄₀ levels and a non-significant increase inAβ₄₂. However, at both 24 and 72 hours there was a two to three-foldincrease in both Aβ₄₀ and Aβ₄₂ in the CSF. Similar results were obtainedusing denaturing gel analysis followed by Aβ Western blotting of pooledCSF. The efflux of Aβ through brain interstitial fluid, which isreflected to some degree by CSF levels, likely accounts for the observedincrease in CSF Aβ.

It is significant that the change in CSF Aβ peptide levels cannot be dueto entry of Mab 266 into the CSF since the levels measured 24 hoursafter injection, which are less than 0.1% plasma levels of Mab 266, areinsufficient to account for the changes. These results suggest Aβpeptide is withdrawn from the brain parenchyma into the CSF by thepresence of the antibody in the bloodstream.

Forms of Aβ peptide which are soluble in PBS or carbonate buffer weremeasured in cerebral cortical homogenates in the same mice which hadbeen injected with Mab 266 and in which the CSF was analyzed asdescribed above. Similar increases in these soluble forms in thecortical homogenates were observed.

EXAMPLE 7 Mab 266 Acts as an Aβ Peptide Sink In Vitro

A dialysis chamber was constructed as an in vitro system to test theability of Mab 266 to act as a sink for Aβ peptide. One mL of human CSFwas placed in the top chamber of a polypropylene tube separated by adialysis membrane with a specified cutoff in the range 10–100 kD from abottom chamber containing 75 μL PBS with or without 1 μg of Mab 266.

It appeared that equilibrium was reached after 3 hours, as determined bysubjecting material in the bottom chamber to acid urea gels followed byWestern blotting for Aβ peptide with 6E10 at various time points Sampleswere denatured in formic acid to a final concentration of 80% (vol/vol)and reduced with β-mercaptoethanol (1%). Samples were electrophoresed(anode to cathode) in a 0.9 M acetic acid running buffer through a 4% to35% polyacrylamide gradient gel containing 6 M urea, 5% (vol/vol)glacial acetic acid, and 2.5% TEMED. The acidic pH of the gel wasneutralized prior to transfer to nitrocellulose. Subsequently, standardWestern blotting techniques were used to identify Aβ. The bands detectedcorrespond to 4 kD.

The amount of Aβ removed from the top chamber was thus determined byELISA analysis of both top and bottom chambers (n=4) after 3 hours. Theresults for various molecular weight cutoffs in the presence and absenceof Mab 266 are shown in FIG. 1. As shown, while only minimal amounts ofAβ peptide crossed the membrane when PBS was placed in the bottomchamber, 50% of the Aβ peptide was sequestered in the bottom chamberwhen Mab 266 was present and the molecular weight cutoff was 25 kD;increasing amounts crossed as the molecular weight cutoff increased to100 kD, when almost 100% of the Aβ peptide was drawn across themembrane.

It was also observed that the anti-N-terminal Aβ antibodies 3D6 and 10D5were able to draw Aβ peptide across the membrane in this system, thoughnot able to sequester Aβ peptide in the assays described in Example 1.These results show that antibodies to the Aβ peptide have sufficientaffinity under these conditions to sequester the peptide inphysiological solutions away from other binding proteins, but that Mabssuch as 266 which are immunoreactive with an epitope in positions 13–28are substantially more efficient and bind with higher affinity.

In similar assays, astrocyte-secreted apoE4 which was purified asdescribed by DeMattos, R. B., et al., J. Biol. Chem. (1998)273:4206–4212; Sun, Y., et al., J. Neurosci. (1998) 18:3261–3272, had asmall by statistically significant effect in increasing the mass of Aβpeptide in the bottom chamber. No apparent affect was observed whenpolyclonal IgG or BSA was substituted for Mab 266.

EXAMPLE 8 Flux of Aβ Peptide into Plasma from the CNS

A. One μg of Aβ₄₀ was dissolved into 5 μL of rat CSF to keep it solubleand was then injected into the subarachnoid space of the cisterna magnaof wild-type Swiss-Webster mice which had previously received IVinjections of either PBS (n=3) or 200 μg of biotinylated Mab 266 (n=3).At different time-points following treatment, Aβ_(Total) in the plasmaof the mice was determined by Aβ ELISA, using 3D6 as the coatingantibody and standards of Aβ mixed with an excess of biotinylated 266.Each plasma sample was spiked with an excess of biotinylated 266 afterremoval from each animal for Aβ detection in the ELISA. In thePBS-injected mice, minimally detectable amounts of the peptide at levelsof 0.15 ng/ml were detected as peak values after 30–60 minutes, afterwhich the levels were essentially zero. In the mice administered Mab266, however, plasma Aβ peptide reached levels 330-fold higher thanthose detected in PBS-injected mice after 60 minutes (approximately 50ng/ml) and reached values of approximately 90 ng/ml after 180 minutes.

B. This procedure was repeated using either 200 μg (n=3) or 600 μg (n=3)injected IV into two-month-old APP^(V717F) mice. Mab 266 was injectedi.v. into 3 month old APP^(V717F)+/+mice with the above doses. Prior toand at different time-points following i.v. injection, the plasmaconcentration of Aβ bound to Mab 266 was determined by RIA. The detailedresults from one illustrative mouse are shown in FIG. 2.

It was found that the concentration of Aβ bound to the monoclonalantibody Mab 266 increased from basal levels of 150 pg/ml to levels ofover 100 ng/ml by four days. By analyzing early time points on thecurve, it was determined that the net rate of entry of Aβ_(Total) intoplasma of the APP^(V717F) Tg mice was 42 pg/ml/minute in the presence ofsaturating levels of the antibody.

The effects of Mab 266 on plasma Aβ levels in both wild type andAPP^(V717F) Tg mice as well as the effects of the antibody on Aβconcentration in CSF show that the presence of circulating Mab 266results in a change in the equilibrium of Aβ flux or transport betweenthe CNS and plasma.

EXAMPLE 9 Mab 266 Effect on Aβ in the Brain

Four month old APP^(V717F)+/+ mice were treated every 2 weeks for 5months with IP injections of saline, Mab 266 (500 μg), or control mouseIgG (100 μg, Pharmigen). The mice were sacrificed at nine months of age,and Aβ deposition in the cortex was determined. The % area covered byAβ-immunoreactivity, as identified with a rabbit pan-Aβ antibody (QCB,Inc.), was quantified in the cortex immediately overlying the dorsalhippocampus as described by Holtzman, D. M., et al., Ann. Neurol. (2000)97:2892–2897. The results are shown in FIG. 3A. At this age, about halfof each group has still not begun to develop Aβ deposition. However, the% of mice with >50% Aβ burden in the cortex was significantly less(P=0.02, Chi-square test) in the 266-treated group. While APP^(V717F)mice can develop large amounts of Aβ deposits by nine months, there isgreat variability with about 50% showing no deposits and about 50%showing substantial deposits. In PBS and IgG treated animals, 6/14 and5/13 mice had greater than 50% of the cortex covered by Aβ staining,while only one of 14 mice treated with Mab 266 had this level ofstaining. Almost 50% of the animals in all groups still had notdeveloped Aβ deposition by 9 months of age. The latter appears to be dueto parental origin of individual mice in our cohort since even thoughall mice studied were confirmed to be APP^(V717F+/+), high levels of Aβdeposition was observed only in mice derived from 4/8 breeding pairs(High pathology litters). Mice derived from the other 4 breeding pairswere virtually free of Aβ deposits (Low pathology litters). Usingparental origin as a co-variate, there was a strong, significant effectof m266 in reducing Aβ deposition (p=0.0082, FIG. 3B).

EXAMPLE 10 Peripherally Injected Mab 266 Does Not Bind to Plagues inAPP^(V717F) Tg Mice

To determine whether Mab 266 injected i.p. over 5 months was bound to Aβin brain, brain sections from 9 month old APP^(V717F+/+) Tg mice whichcontained Aβ deposits and had been treated with either Mab 266, saline,or control IgG were utilized. Tissue processing and immunostaining wasperformed as described (Bales, K. R., et al., Nature Genet. (1997)17:263–264). Tissue from all groups of animals was incubated withfluorescein-labeled anti-mouse IgG (Vector, Inc.) and then examinedunder a fluorescent microscope. No specific staining of Aβ deposits wasseen in any of the groups. In contrast, when applying Mab 266 tosections prior to incubation of the sections with anti-mouse IgG, Aβdeposits were clearly detected.

EXAMPLE 11 Effect of Administration of Antibody 266 on Cognition in24-Month Old Transgenic, Hemizygous PDAPP Mice

Sixteen hemizygous transgenic mice (APP^(V717F)) were used. The micewere approximately 24 months old at the start of the study. Allinjections were intraperitoneal (i.p.). Half the mice received weeklyinjections of phosphate buffered saline (PBS, “Control”) and the otherhalf received 500 micrograms of mouse antibody 266 dissolved in PBS.Injections were made over a period of seven weeks (42 days) for a totalof six injections. Three days following the last injection, the behaviorof the animals was assessed using an object recognition task,essentially as described in J. -C. Dodart, et al., BehavioralNeuroscience, 113 (5) 982–990 (1999). A recognition index(T_(B)×100)/(T_(B)−T_(A)) was calculated. Results are shown below inTable 1.

TABLE 1 Descriptive statistics for recognition index Recognition Index(minutes) Standard Standard N Mean Deviation Error Control (PBS) 871.2** 8.80 3.11 Antibody 266 8 54.35  7.43 2.62 **p = 0.0010

Administration of 500 micrograms of antibody 266 weekly to 24 month old,hemizygous, transgenic mice was associated with a significant change inbehavior. Antibody treated transgenic mice had recognition indices whichwere similar to wildtype control animals [J. -C. Dodart, et al]. Thedifference in the recognition index was statistically significant at the0.001 probability level. The increased recognition index is anindication that treatment with an antibody that binds to the betaamyloid peptide in the region of amino acids 13–28 will reverse thebehavioral impairments that had been documented in this mouse model ofAlzheimer's Disease. Therefore, the administration of antibodies thatbind beta amyloid peptide in the region of amino acids 13–28 will treatdiseases such as Alzheimer's disease and Down's syndrome and will haltthe cognitive decline typically associated with disease progression.

The amyloid burden (% area covered by immunoreactive material afterstaining with anti-Aβ antibodies 3D6 or 21F12) was quantified in thecortex immediately overlying the hippocampus including areas of thecingulate and parietal cortex from the brains of the 24 month-oldanimals treated with mouse antibody 266 for seven weeks, as describedabove. The results are presented in the table below. The differencesbetween the treatment groups are not statistically significant.

TABLE 2 Amyloid plaque burden in APP^(V717F+/−) mice following treatmentwith mouse 266 anti-Aβ antibody Plague Burden (%) Using 3D6 Using 21F12Standard Standard N Mean Error Mean Error Control (PBS) 7 44.3 5.93 0.770.14 Antibody 266 8 38.0 2.96 0.93 0.11

For these very old animals, treatment with mouse antibody 266 did notresult in a significantly different amyloid burden compared with thePBS-treated group, measured using either 3D6 or using 21F12.Furthermore, the Aβ burden was substantially greater and significantlyincreased compared with the amyloid burden in younger animals (seebelow) who were not able to discriminate a novel object from a familiarone in the object recognition task. Most surprisingly, these resultsdemonstrate that anti-Aβ antibodies can reverse cognitive deficitswithout the need to reduce amyloid burden per se.

After 7 weeks of treatment, the recognition index of the m266-treatedgroup was not significantly different than what would be expected for awild type cohort of 24 month old mice! This indicates a completereversal of cognitive decline in these transgenic animals.

EXAMPLE 12 Effect of Administration of Antibody 266 on Cognition inYoung Transgenic, Hemizygous PDAPP Mice

Fifty-four (54) homozygous, transgenic mice (APP^(V717F)) were used.Twenty-three (23) mice were approximately two months old at the start ofthe study. The remaining mice were approximately four months old at thestart of the study. The duration of treatment was five months. Thus, atstudy termination, the mice were either approximately seven (7) monthsold or approximately nine (9) months old.

All injections were intraperitoneal (i.p.). Each mouse in “PBS” controlgroups received a weekly injection of phosphate buffered saline (PBS;200 μL). Each mouse in the “IgG” control groups received a weeklyinjection of IgG1κ1 isotype control (100 μg/mouse/week). Each mouse inthe “High Dose” groups received a weekly injection of 500 microgram ofantibody 266 dissolved in PBS (“HD”). Each mouse in the “Low Dose” groupreceived a weekly injection of 100 micrograms of antibody 266 dissolvedin PBS (“LD”). Three days following the last injection, the behavior ofthe animals was assessed using an object recognition task, as describedin Example 10 above, and a discrimination index was calculated as thedifference between the time spent on a novel object and the time spenton a familiar object. Results are shown below in Table 3. The data aregrouped by the age of the mice at the end of the study.

TABLE 3 Descriptive statistics for discrimination index DiscriminationIndex (minutes) Standard Standard N Mean Deviation Error 7 months oldPBS 7 2.12 4.22 1.59 IgG 8 0.81 3.64 1.29 HD 8 10.04* 6.52 2.30 9 monthsold PBS 7 1.87 3.54 1.34 IgG 8 0.96 3.51 1.24 LD 8 10.75* 6.44 2.28 HD 812.06*** 7.82 2.76 *p < 0.05 ***p < 0.0001

Taken together these data support the conclusion that administration ofantibody 266, an antibody directed against the central domain of AP,attenuates plaque deposition in 7–9 month old APP^(V717F) transgenicmice, as well as reverses the behavioral impairments previouslycharacterized. Treatment of patients with an antibody directed againstthe central domain of the Aβ peptide will inhibit or prevent cognitivedecline typically associated with disease progression, and will reverseit.

The discrimination index for treated animals was not significantlydifferent than what would be expected for wild type mice of the sameage. Thus, just as in older animals (Example 11), treatment with m266completely reversed cognitive decline in these younger transgenicanimals.

EXAMPLE 13 Synthesis of Humanized Antibody 266

Cells and antibodies. Mouse myeloma cell line Sp2/0 was obtained fromATCC (Manassas, Va.) and maintained in DME medium containing 10% FBS(Cat # SH32661.03, HyClone, Logan, Utah) in a 37° C. CO₂ incubator.Mouse 266 hybridoma cells were first grown in RPMI-1640 mediumcontaining 10% FBS (HyClone), 10 mM HEPES, 2 mM glutamine, 0.1 mMnon-essential amino acids, 1 mM sodium pyruvate, 25 μg/ml gentamicin,and then expanded in serum-free media (Hybridoma SFM, Cat # 12045–076,Life Technologies, Rockville, Md.) containing 2% low Ig FBS (Cat #30151.03, HyClone) to a 2.5 liter volume in roller bottles. Mousemonoclonal antibody 266 (Mu266) was purified from the culturesupernatant by affinity chromatography using a protein-G Sepharosecolumn. Biotinylated Mu266 was prepared using EZ-LinkSulfo-NHS-LC-LC-Biotin (Cat # 21338ZZ, Pierce, Rockford, Ill.).

Cloning of variable region cDNAs. Total RNA was extracted fromapproximately 10⁷ hybridoma cells using TRIzol reagent (LifeTechnologies) and poly(A)⁺ RNA was isolated with the PolyATract mRNAIsolation System (Promega, Madison, Wis.) according to the suppliers'protocols. Double-stranded cDNA was synthesized using the SMART™ RACEcDNA Amplification Kit (Clontech, Palo Alto, Calif.) following thesupplier's protocol. The variable region cDNAs for the light and heavychains were amplified by polymerase chain reaction (PCR) using 3′primers that anneal respectively to the mouse kappa and gamma chainconstant regions, and a 5′ universal primer provided in the SMARTTMRACEcDNA Amplification Kit. For VL PCR, the 3′ primer has the sequence:

5′-TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC-3′ [SEQ ID NO:13] withresidues 17–46 hybridizing to the mouse Ck region. For VH PCR, the 3′primers have the degenerate sequences:

                                A       G   T5′-TATAGAGCTCAAGCTTCCACTGGATAGACCGATGGGGCTGTCGTTTTGGC-3′ [SEQ ID NO:14]                                Twith residues 17–50 hybridizing to mouse gamma chain CHI. The VL and VHcDNAs were subcloned into pCR4Blunt-TOPO vector (Invitrogen, Carlsbad,Calif.) for sequence determination. DNA sequencing was carried out byPCR cycle sequencing reactions with fluorescent dideoxy chainterminators (Applied Biosystems, Foster City, Calif.) according to themanufacturer's instruction. The sequencing reactions were analyzed on aModel 377 DNA Sequencer (Applied Biosystems).

Construction of humanized 266 (Hu266) variable regions. Humanization ofthe mouse antibody V regions was carried out as outlined by Queen et al.[Proc. Natl. Acad. Sci. USA 86:10029–10033 (1988)]. The human V regionframework used as an acceptor for Mu266 CDRs was chosen based onsequence homology. The computer programs ABMOD and ENCAD [Levitt, M., J.Mol. Biol. 168:595–620 (1983)] were used to construct a molecular modelof the variable regions. Amino acids in the humanized V regions thatwere predicted to have contact with CDRs were substituted with thecorresponding residues of Mu266. This was done at residues 46, 47, 49,and 98 in the heavy chain and at residue 51 in the light chain. Theamino acids in the humanized V region that were found to be rare in thesame V-region subgroup were changed to the consensus amino acids toeliminate potential immunogenicity. This was done at residues 42 and 44in the light chain.

The light and heavy chain variable region genes were constructed andamplified using eight overlapping synthetic oligonucleotides ranging inlength from approximately 65 to 80 bases [He, X. Y., et al., J. Immunol.160: 029–1035 (1998)]. The oligonucleotides were annealed pairwise andextended with the Klenow fragment of DNA polymerase I, yielding fourdouble-stranded fragments. The resulting fragments were denatured,annealed pairwise, and extended with Klenow, yielding two fragments.These fragments were denatured, annealed pairwise, and extended onceagain, yielding a full-length gene. The resulting product was amplifiedby PCR using the Expand High Fidelity PCR System (Roche MolecularBiochemicals, Indianapolis, Ind.). The PCR-amplified fragments weregel-purified and cloned into pCR4Blunt-TOPO vector. After sequenceconfirmation, the VL and VH genes were digested with MIuI and XbaI,gel-purified, and subcloned respectively into vectors for expression oflight and heavy chains to make pVk-Hu266 and pVg1-Hu266 (see FIGS. 6 and7, respectively, herein) [Co, M. S., et al., J. Immunol. 148:1149–1154(1992)]. The mature humanized 266 antibody expressed from these plasmidshas the light chain of SEQ ID NO:11 and the heavy chain of SEQ ID NO:12.

Stable transfection. Stable transfection into mouse myeloma cell lineSp2/0 was accomplished by electroporation using a Gene Pulser apparatus(BioRad, Hercules, Calif.) at 360 V and 25 μF as described (Co et al.,1992). Before transfection, pVk-Hu266 and pVg1-Hu266 plasmid DNAs werelinearized using FspI. Approximately 10⁷ Sp2/0 cells were transfectedwith 20 g of pVk-Hu266 and 40 μg of pVg1-Hu266. The transfected cellswere suspended in DME medium containing 10% FBS and plated into several96-well plates. After 48 hr, selection media (DME medium containing 10%FBS, HT media supplement, 0.3 mg/ml xanthine and 1 μg/ml mycophenolicacid) was applied. Approximately 10 days after the initiation of theselection, culture supernatants were assayed for antibody production byELISA as shown below. High yielding clones were expanded in DME mediumcontaining 10% FBS and further analyzed for antibody expression.Selected clones were then adapted to growth in Hybridoma SFM.

Measurement of antibody expression by ELISA. Wells of a 96-well ELISAplate (Nunc-Immuno plate, Cat # 439454, NalgeNunc, Naperville, Ill.)were coated with 100 μl of 1 μg/ml goat anti-human IgG, Fcγ fragmentspecific, polyclonal antibodies (Cat # 109-005-098, JacksonImmunoResearch, West Grove, Pa.) in 0.2 M sodium carbonate-bicarbonatebuffer (pH 9.4) overnight at 4° C. After washing with Washing Buffer(PBS containing 0.1% Tween 20), wells were blocked with 400 μl ofSuperblock Blocking Buffer (Cat # 37535, Pierce) for 30 min and thenwashed with Washing Buffer. Samples containing Hu266 were appropriatelydiluted in ELISA Buffer (PBS containing 1% BSA and 0.1% Tween 20) andapplied to ELISA plates (100 μl per well). As a standard, humanizedanti-CD33 IgG1 monoclonal antibody HuM195 (Co, et al., 1992, above) wasused. The ELISA plate was incubated for 2 hr at room temperature and thewells were washed with Wash Buffer. Then, 100 μl of 1/1,000-dilutedHRP-conjugated goat antihuman kappa polyclonal antibodies (Cat #1050-05, Southern Biotechnology, Birmingham, Ala.) in ELISA Buffer wasapplied to each well. After incubating for 1 hr at room temperature andwashing with Wash Buffer, 100 μl of ABTS substrate (Cat #s 507602 and506502, Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was addedto each well. Color development was stopped by adding 100 μl of 2%oxalic acid per well. Absorbance was read at 415 nm using an OPTImaxmicroplate reader (Molecular Devices, Menlo Park, Calif.).

Purification of Hu266. One of the high Hu266-expressing Sp2/0 stabletransfectants (clone 1D9) was adapted to growth in Hybridoma SFM andexpanded to 2 liter in roller bottles. Spent culture supernatant washarvested when cell viability reached 10% or below and loaded onto aprotein-A Sepharose column. The column was washed with PBS before theantibody was eluted with 0.1 M glycine-HCl (pH 2.5), 0.1 M NaCl. Theeluted protein was dialyzed against 3 changes of 2 liter PBS andfiltered through a 0.2 μm filter prior to storage at 4° C. Antibodyconcentration was determined by measuring absorbance at 280 nm (1mg/ml=1.4 A₂₈₀). SDS-PAGE in Tris-glycine buffer was performed accordingto standard procedures on a 4–20% gradient gel (Cat # EC6025, Novex, SanDiego, Calif.). Purified humanized 266 antibody is reduced and run on anSDSPAGE gel. The whole antibody shows two bands of approximate molecularweights 25 kDa and 50 kDa. These results are consistent with themolecular weights of the light chain and heavy chain or heavy chainfragment calculated from their amino acid compositions.

EXAMPLE 14 In Vitro Binding Properties of Humanized 266 Antibody

The binding efficacy of humanized 266 antibody, synthesized and purifiedas described above, was compared with the mouse 266 antibody usingbiotinylated mouse 266 antibody in a comparative ELISA. Wells of a96-well ELISA plate (Nunc-immuno plate, Cat # 439454, NalgeNunc) werecoated with 100 μl of β-amyloid peptide (1–42) conjugated to BSA in 0.2M sodium carbonate/bicarbonate buffer (pH 9.4) (10 μg/mL) overnight at4° C. The Aβ₁₋₄₂-BSA conjugate was prepared by dissolving 7.5 mg ofAβ₁₋₄₂-Cys₄₃ (C-terminal cysteine Aβ₁₋₄₂, AnaSpec) in 500 μL ofdimethylsulfoxide, and then immediately adding 1,500 μL of distilledwater. Two (2) milligrams of maleimide-activated bovine serum albumin(Pierce) was dissolved in 200 μL of distilled water. The two solutionswere combined, thoroughly mixed, and allowed to stand at roomtemperature for two (2) hours. A gel chromatography column was used toseparate unreacted peptide from Aβ₁₋₄₂-Cys-BSA conjugate.

After washing the wells with phosphate buffered saline (PBS) containing0.1% Tween 20 (Washing Buffer) using an ELISA plate washer, the wellswere blocked by adding 300 μL of SuperBlock reagent (Pierce) per well.After 30 minutes of blocking, the wells were washed Washing Buffer andexcess liquid was removed.

A mixture of biotinylated Mu266 (0.3 μg/ml final concentration) andcompetitor antibody (Mu266 or Hu266; starting at 750 μg/ml finalconcentration and serial 3-fold dilutions) in ELISA Buffer were added intriplicate in a final volume of 100 μl per well. As a no-competitorcontrol, 100 μl of 0.3 μg/ml biotinylated Mu266 was added. As abackground control, 100 μl of ELISA Buffer was added. The ELISA platewas incubated at room temperature for 90 min. After washing the wellswith Washing Buffer, 100 μl of 1 μg/ml HRP-conjugated streptavidin (Cat# 21124, Pierce) was added to each well. The plate was incubated at roomtemperature for 30 min and washed with Washing Buffer. For colordevelopment, 100 μl/well of ABTS Peroxidase Substrate (Kirkegaard &Perry Laboratories) was added. Color development was stopped by adding100 μl/well of 2% oxalic acid. Absorbance was read at 415 nm. Theabsorbances were plotted against the log of the competitorconcentration, curves were fit to the data points (using Prism) and theIC50 was determined for each antibody using methods well-known in theart.

The mean IC50 for mouse 266 was 4.7 μg/mL (three separate experiments,standard deviation=1.3 μg/mL) and for humanized 266 was 7.5 μg/mL (threeseparate experiments, standard deviation=1.1 μg/mL). A second set ofthree experiments were carried out, essentially as described above, andthe mean IC50 for mouse 266 was determined to be 3.87 μg/mL (SD=0.12μg/mL) and for human 266, the IC50 was determined to be 4.0 μg/mL(SD=0.5 μg/mL). On the basis of these results, we conclude thathumanized 266 has binding properties that are very similar to those ofthe mouse antibody 266. Therefore, we expect that humanized 266 has verysimilar in vitro and in vivo activities compared with mouse 266 and willexhibit in humans the same effects demonstrated with mouse 266 in mice.

EXAMPLE 15 In Vitro Binding Properties of Mouse Antibodies 266 and 4G8

Antibody affinity (KD=Kd/Ka) was determined using a BIAcore biosensor2000 and data analyzed with BIAevaluation (v. 3.1) software. A captureantibody (rabbit anti-mouse) was coupled via free amine groups tocarboxyl groups on flow cell 2 of a biosensor chip (CM5) usingN-ethyl-N-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide(EDC/NHS). A non-specific rabbit IgG was coupled to flow cell 1 as abackground control Monoclonal antibodies were captured to yield 300resonance units (RU). Amyloid-beta 1–40 or 1–42 (BiosourceInternational, Inc.) was then flowed over the chip at decreasingconcentrations (1000 to 0.1 times KD). To regenerate the chip, boundanti-Aβ antibody was eluted from the chip using a wash with glycine-HCl(pH 2). A control injection containing no amyloid-beta served as acontrol for baseline subtraction. Sensorgrams demonstrating associationand dissociation phases were analyzed to determine Kd and Ka. Using thismethod, the affinity of mouse antibody 266 for both Aβ₁₋₄₀ and forAβ₁₋₄₂ was found to be 4 pM. The affinity of 4G8 for Aβ₁₋₄₀ was 23 nMand for Aβ₁₋₄₂ was 24 nM. Despite a 6000-fold difference in affinitiesfor Aβ, both 266 and 4G8, which bind to epitopes between amino acids 13and 28 of Aβ, effectively sequester Aβ from human CSF. Therefore, thelocation of the epitope is paramount, rather than binding affinity, indetermining the ability of an antibody to sequester Aβ and to providethe beneficial and surprising advantages of the present invention.

EXAMPLE 16 Epitope Mapping of Mouse Antibody 266 Using BiacoreMethodolgy and Soluble Peptides

The BIAcore is an automated biosensor system for measuring molecularinteractions [Karlsson R., et al. J. Immunol. Methods 145:229–240(1991)]. The advantage of the BIAcore over other binding assays is thatbinding of the antigen can be measured without having to label orimmobilize the antigen (i.e. the antigen maintains a more nativeconformation). The BIAcore methodology was used to assess the binding ofvarious amyloid-beta peptide fragments to mouse antibody 266,essentially as described above in Example 12, except that all dilutionswere made with HEPES buffered saline containing Tween 20, a variety offragments of A□ (BioSource International) were injected, and a singleconcentration of each fragment was injected (440 nM).

Amyloid beta fragments 1–28, 12–28, 17–28 and 16–25 bound to mouseantibody 266, while Aβ fragments 1–20, 10–20, and 22–35 did not bind.Fragments 1–20, 10–20 and 22–35 bound to other MAbs with known epitopespecificity for those regions of Aβ. Using this methodology, the bindingepitope for the mouse antibody 266 appears to be between amino acids 17and 25 of Aβ. Since binding usually occurs with at least 3 residues ofthe epitope present, one could further infer that the epitope iscontained within residues 19–23.

EXAMPLE 17 In Vitro Binding Properties of Humanized Antibody 266

The affinity (KD=Kd/Ka) of humanized 266 antibody, synthesized andpurified as described above, was determined essentially as describedabove in Example 15. Using this method, the affinity of humanized 266for Aβ ₁₋₄₂ was found to be 4 pM.

1. A humanized antibody comprising: (a) a light chain comprising a lightchain variable region comprising the following sequence: (SEQ ID NO:7)1                 5                  10 Asp Xaa Val Met Thr Gln Xaa ProLeu Ser Leu Pro Val      15                  20                  25 XaaXaa Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser             30                  35 Gln Ser Leu Xaa Tyr Ser Asp Gly AsnAla Tyr Leu His  40                  45                  50 Trp Phe LeuGln Lys Pro Gly Gln Ser Pro Xaa Leu Leu         55                  60                  65 Ile Tyr Lys Val SerAsn Arg Phe Ser Gly Val Pro Asp                  70                  75Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu     80                  85                  90 Lys Ile Ser Arg Val GluAla Glu Asp Xaa Gly Val Tyr              95                 100 Tyr CysSer Gln Ser Thr His Val Pro Trp Thr Phe Gly 105                 110 XaaGly Thr Xaa Xaa Glu Ile Lys Arg

wherein: Xaa at position 2 is Vle or Ile; Xaa at position 7 is Ser orThr; Xaa at position 14 is Thr or Ser; Xaa at position 15 is Leu or Pro;Xaa at position 30 is Ile or Val; Xaa at position 50 is Arg, Gln, orLys; Xaa at position 88 is Val or Leu; Xaa at position 105 is Gln orGly; Xaa at position 108 is Lys or Arg; and Xaa at position 109 is Valor Leu; and (b) a heavy chain comprising a heavy chain variable regioncomprising the following sequence: (SEQ ID NO:8)1                5                   10 Xaa Val Gln Leu Val Glu Xaa GlyGly Gly Leu Val Gln      15                  20                  25 ProGly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly             30                  35 Phe Thr Phe Ser Arg Tyr Ser Met SerTrp Val Arg Gln  40                  45                  50 Ala Pro GlyLys Gly Leu Xaa Leu Val Ala Gln Ile Asn         55                  60                  65 Ser Val Gly Asn SerThr Tyr Tyr Pro Asp Xaa Val Lys                  70                  75Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa Xaa Asn Thr     80                  85                  90 Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Xaa Asp Thr              95                 100 Ala ValTyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln 105                 100 GlyThr Xaa Val Thr Val Ser Ser

wherein: Xaa at position 1 is Glu or Gln; Xaa at position 7 is Ser orLeu; Xaa at position 46 is Glu, Val, Asp, or Ser; Xaa at position 63 isThr or Ser; Xaa at position 75 is Ala, Ser, Val or Thr; Xaa at position76 is Lys or Arg; Xaa at position 89 is Glu or Asp; and Xaa at position107 is Leu or Thr and wherein the humanized antibody binds to an abetapeptide.
 2. The humanized antibody of claim 1 comprising a light chainvariable region of the sequence given by SEQ ID NO:9 and a heavy chainvariable region given by SEQ ID NO:10.
 3. The humanized antibody ofclaim 1 comprising a light chain of the sequence given by SEQ ID NO:11and a heavy chain of the sequence given by SEQ ID NO:12.
 4. Thehumanized antibody of claim 1 that is an IgG1 immunoglobulin isotype. 5.The humanized antibody of claim 1, wherein the antibody is produced in ahost cell selected from the group consisting of a meyeloma cell, achinese hamster ovary cell, a syrian hamster ovary cell, and a humanembryonic kidney cell.
 6. A pharmaceutical composition that comprisesthe humanized antibody of claim 1, and a pharmaceutically acceptableexcipient.
 7. A pharmaceutical composition that comprises the humanizedantibody of claim 3, and a pharmaceutically acceptable excipient.
 8. Thehumanized antibody of claim 1, wherein Xaa at position 30 of the lightchain variable region is lie and wherein Xaa at position 63 of the heavychain variable region is Thr.
 9. The humanized antibody of claim 2 thatis an IgG1 immunoglobulin isotype.
 10. The humanized antibody of claim 3that is an IgG1 immunoglobulin isotype.
 11. The humanized antibody ofclaim 2, wherein the antibody is produced in a chinese hamster ovarycell.
 12. The humanized antibody of claim 3, wherein the antibody isproduced in a chinese hamster ovary cell.